Transcript Malpighian Tubules

Insect Biochemistry
- Excretion
Kuang-Hui Lu
Department of Entomology
National Chung Hsing University
CONTENTS
Introduction
The Malpighian tubules
Ultrastructure of Malpighian tubule cells
Formation of primary urine in Malpighian
tubules
A proton pump is the driving mechanism for
urine formation
Selective reabsorption in the hindgut
The role of the excretory system in
maintaining homeostasis
Cryptonephridial systems
Introduction
Excretion: any process that eliminates the
interaction of harmful substances with
cells and tissues.
– eliminate nitrogenous metabolites
– maintain ions and water balance
– remove ingested chemicals – e.g.
allelochemicals
Introduction
Excretory organs of insects
– Malpighian tubules – collect a filtrate from the
hemolymph and pass this primary urine to the hindgut.
– Hindgut – secrete additional components into the
secreta, and reabsorbe some substances into the
hemolymph.
Introduction
The major function of excretory system is
to maintain the internal environment of an
organism by separating and eliminating
metabolic wastes and other toxic
substances.
Because metabolic wastes are often
dissolved in water, excretory processes
are also closely associated with
osmoregulation and the maintenance of
water balance.
Fig. A generalized scheme of excretion showing the collection of fluid in the
Malpighian tubules, and extensive reabsorption of water, K+, and useful
substances from the hindgut, primarily the rectum.
Malpighian Tubules
Malpighian tubules are primary excretory
organs of insects
Malpighian tubules are long, tubular structures,
usually arising at the junction of the mid- and
hindgut and terminating blindly in the hemocoel.
The tubules vary in number from 2 to more than
100 in various insect species.
Tracheal connections to Malpighian tubules are
numerous.
A small spiral muscle frequently runs along the
surface of a tubule. (next slide)
Malpighian Tubules
The Malpighian tubules arise during embryogenesis as
evaginations of the gut, usually originating at the junction of
the midgut and hindgut.
The tubule walls consist of a single cell layer of epithelial
cells and are differentiated by structure and function along
the length of the tubule.
Malpighian Tubules
The process of excretion is a two-step process,
with much of the fluid that is taken up by the
tubules resorbed by the hindgut before is passes
out of the body.
Ultrastructure of
Malpighian Tubule Cells
A single layer of epithelial cells surrounds
the lumen of a tubule.
Several different cell types have been
identified, but their specific functions have
not been elucidated in many cases.
Type 1 (or principal tubule cells) – tubule
cells have a brush border of microvilli on
the apical surface. (next slide)
Transport of Substances Through
the Malpighian Tubule Cells
The primary urine formed in the lumen of the Malpighian
tubules is a filtrate of the hemolymph, and it contains
most of the small ions and molecules that occur in the
hemolymph.
Transport of Substances Through
the Malpighian Tubule Cells
The urine:hemolymph concentration ratio
for many of the filtered substances
approaches unity, indicating passive
movement across the tubule cell
membranes.
But some components are actively
secreted and their urine:hemolymph ratio
is always greater than one.
Primary Urine Formation
Urine formation in Malpighian tubules mainly
relies on a proton pump in the apical membrane
of Malpighian tubule cells that actively secretes
protons (H+) into the tubule lumen against an
electrochemical gradient.
The pump causes the tubule lumen to become
positive to the hemolymph, and creates highly
variable gradients in pH across the apical
membrane of principal cells.
The proton gradient provides the energy for an
antiporter mechanism that exchanges K+ for H+
across the apical membrane. (next slide)
Primary Urine Formation
Secretion of cations (H+, Na+, and K+) across the
apical membrane appears to be electrically
coupled with Cl- transport in the basolateral
membrane of tubule cells. (next slide)
The formation of urine volume is highly dependent
on the K+ concentration in hemolymph or saline.
(next slide)
The process driven by the proton pump has been
called a standing gradient process.
Additional processes might be involved in
substances interring the tubule lumen. (next slide)
Hormonal Control of
Urine Formation
The rates of urine formation and ion secretion
are controlled by diuretic hormones and certain
non-peptide compounds, such as 5hydroxytryptamine (5-HT or serotonin)
The diuretic neuropeptides isolated from insects
fall into one of two hormone families:
– Corticotropin-releasing factor (CRF)-related peptides:
range in size from 30-46 amino acids; has
approximately 30% sequence homology with the CRF
family of vertebrate peptides.
– Insect kinins: small peptides of between 6 and 15
amino acids
Urine Formation
The primary urine formed by the Malpighian
tubules is isosmotic or sometimes slightly
hyposmotic to the hemolymph.
Malpighian tubules are not capable of
producing primary urine that is appreciably
hyperosmotic to the hemolymph.
The hindgut proceeds to concentrate waste
products by reabsorbing water and useful
substances.
The Bioassay of Malpighian Tubule
Function Devised by Ramsay
By analyzing the primary urine formed in the droplets, it was discovered that it
was isosmotic with the hemolymph, but with potassium concentrations up to 20
times higher.
Arrangement for Experimental
Perfusion of an Isolated Tubule
The Cumulative Formation of
Primary Urine by an Isolated Tubule
Anatomical Specialization of
Hindgut Epithelial Cells
The hindgut is the second system that completes
the excretion process by
– selectively reabsorbing some substances into the
hemolymph
– leaving others in the lumen
– actively secreting some substance into the hindgut lumen
The rectal cuticular lining has greater permeability
than the cuticular lining on foregut cells.
The epithelial cells of the hindgut are specialized for
both active secretion and active reabsorption.
Anatomical Specialization of
Hindgut Epithelial Cells
Rectal cells (or rectal pad cells, rectal papillae
cells) – a group cells in the rectum that have special
modifications for reabsorption. (next slide)
In Diptera, the cells of a rectal papilla are large,
usually cuboidal cells that surround a central
channel in the papilla that opens into the
hemolymph space through a valve. (next slide).
A Rectal Cell and Its Ion Transport
The rectum consists of the enlarged posterior-most
section of the hindgut, often containing specialized
structures called papillae or rectal pads that are enlarged
epithelial cells.
The rectum transports water and ions from the material
within the gut lumen into the hemolymph.
Secretion and Reabsorption in
the Ileum
The ileum is the most anterior part of the
hindgut, occurring just posterior to the origin of
the Malpighian tubules in most insects.
In locust S. gregaria, the ileum is a major site
for
– isosmotic fluid reabsorption
– active Na+ and Cl- reabsorption
– active secretion of proline as an energy source
Secretion and Reabsorption in
the Ileum
The driving mechanism for ion and water
reabsorption in the ileum is an electrogenic Clpump.
A neuropeptide, the ion transport peptide (ITP)
stimulates Na+, Cl- and water reabsorption, and
promotes passive reabsorption of K+ by
electrical coupling.
The ileum plays a major role in acid-base
balance by secretion of H+ into the lumen,
formation of NH4+, and reabsorption of HCO3-.
Reabsorption in the Rectum
The rectum is the final and major site for
reabsorption of ions, water, and nutrients.
It is capable of reabsorbing fluid against strong
osmotic gradients, ultimately producing a very
concentrated hyperosmotic excreta in many
insects.
The driving mechanism for cation and water
reabsorption, as in the ileum, is an electrogenic
Cl- pump under the influence of a neuropeptide
hormone, chloride transport stimulating
hormone (CTSH), from the corpora cardiaca.
Fig. Ions are transported in and out of locust rectal cell by
numerous mechanisms.
Electrolyte Homeostasis
In mosquito A. aegypti, feeding on a blood meal
stimulates the release of mosquito natriuretic peptide
(MNP) from the CC, and cAMP is produced and acts
selectively to open Na+ channels in the basolateral
membrane of the Malpighian tubule cells.
Movement of water into tubule cells follows the
osmotic gradient.
The ion flex generated by MNP and cAMP is
specifically an increase in secretion of Na+. K+
movement is not influenced.
The Cl- load from the blood meal move from
hemolymph to tubule lumen in a passive transport
pathway between the cells (paracellular pathway).
Electrolyte Homeostasis
Larval A. aegypti live in fresh water, and in
response to an increase in salinity
– Secrete 5-hydroxytryptamine (serotonin) into the
hemolymph
– Increase cAMP in the Malpighian tubules
– Serotonin and cAMP stimulate fluid and ion (Na+
and K+) secretion rates in the tubules, but urine is
not concentrated with respect to the ions
Electrolyte Homeostasis
Beyenbach (1995) has reviewed three potential
physiological processes through which A. aegypti
may regulate rates of ion and fluid excretion
– The proton pump that supplies energy for Na+ and K+
secretion to the tubule lumen
– The resistance Rc across the tubule cells that control
ion channels in the basolateral membrane
– The resistance of the passive transport pathway for Clmovement
Water Homeostasis
Water excretion and retention are
regulated by hormones.
– Diuretic hormones promote fluid formation and
rapid excretion by the Malpighian tubules
Corticotropin-releasing factor (CRF)-related
peptides: range in size from 30-46 amino acids.
Insect kinins: small peptides of between 6 and 15
amino acids.
– Antidiuretic hormones act on the hindgut and
promote water reabsorption
Chloride transport-stimulating hormone (CTSH)
Ion transport peptide (ITP)
A Filter Chamber
In some Homopterans that feed exclusively on
plant juices containing low concentrations of
nutrients, the digestive tract forms an
arrangement known as a filter chamber.
Acid-Base Homeostasis
The excretory system is important in
maintaining the acid-base balance of
body fluids and tissues.
Acid-base regulation in S. gregaria
– Secretion of H+ and formation of NH4+ in the
ileum is a principal mechanism for excreting
excess acid equivalents.
– The ileum is a major site of ammoniagenesis
in locusts in which hindgut cells specifically
metabolize amino acids and glucose for
energy.
Acid-Base Homeostasis
Excretion of total ammonia nitrogen
serves several functions in locusts
– Ammonium urate (i.e. NH3 reacts with uric
acid) allows the insect to conserve Na+
– Conversion of NH3 to NH4+ in the ileal cells is
equivalent to removal of H+
– Excretion of NH3 by locusts conserves water
– Increases nitrogen excretion by 25% more
than excretion of only Na- or K-urate.
Nitrogen Homeostasis
The metabolism of proteins and nucleic acids
produce ammonia.
Some of this ammonia can be recycled into
amino acid synthesis by the formation of
glutamate from a-ketoglutarate and glutamine
from glutamate.
The excess ammonia that remains is highly
toxic unless it is diluted with water.
High levels of ammonia can interrupt nervous
transmission by substituting for necessary
potassium and can also alter carbohydrate and
lipid metabolism.
The Incorporation of Ammonia for
the Synthesis of Amino Acids
Nitrogen Homeostasis
Organisms must have excretory systems to
avoid the toxic accumulation of ammonia.
Because ammonia is very soluble in water, its
concentrations have to be maintained below
levels that are toxic.
Most terrestrial organisms have taken the
pathway of the incorporation of the nitrogen into
either urea or uric acid, which can be
concentrated in body fluid to a much greater
extent than can ammonia and require less
water for dilution.
Excretory Molecules that
Incorporate Nitrogen
Nitrogen Homeostasis
In insects, the need for water conservation may
have been the driving force for the incorporation
of their nitrogen wastes into uric acid.
The fat body is the primary site for uric acid
synthesis.
Uric acid does not dissolve well in water and
therefore fails to reach toxic levels in body fluids,
so it requires about 50 times less water to dilute
than does ammonia.
Insolubility of uric acid in water allows it to be
excreted in a dry form without having a significant
effect on water balance.
Nitrogen Homeostasis
Insects pay a high price for the benefits
they derive from employing uric acid as a
way to excrete nitrogen and still maintain
a positive water balance.
– The synthesis of uric acid results loss of
several carbon atoms.
– Eight ATP are required to first make the
intermediary metabolite, inosine
monophosphate (IMP)
Xanthine
dehygrogenase
Fig. Pathway for the synthesis of uric acid from
nucleic acids and protein.
One Way to Account for the Release
of Urea by Some Insects
Release of Ammonia by the
Deamination of Amino Acids
e.g. blowfly larvae and some cockroaches and locusts.
Storage Excretion
Because uric acid is so insoluble, it can be
easily stored without it interacting with
other physiological processes.
– Some cockroaches accumulate up to 10% of
their dry weight in uric acid stored in
specialized urate cells in the fat body, which
can be utilized during periods of dietary stress.
– In some lepidoptera, the fat body shifts from
excretion of uric acid to its storage during the
last larval instar.
A Cryptonephridial Complex
Many families of Coleoptera, Lepidoptera and
some saw-fly larvae, that live under extremely
dry conditions, the ends of the Malpighian
tubules do not lie free in the hemocoel.
Instead, the terminal segments of the tubules
are closely associated with the wall of the
rectum in what is called a cryptonephridial
complex. (next slide)
It appears to be an arrangement that enables
very efficient conservation of water.
A Cryptonephridial Complex
The cryptonephridial complex is found in most
lepidopteran larvae and many coleopterans.
The cryptonephridial complex performs two
functions:
– Resorb water from the hindgut very efficiently. (next
slide)
– In some insects is able to absorb atmospheric water
from the humidity in the hindgut.
Fig. An SEM photo of the small muscle (arrow) that often spirals
along the length of a Malpighian tubule of some insects.
A Cross Section through the Primary
Type of Malpighian Tubule
Fig. The general structure of a Malpighian tubule cell from the proximal tubule
segment of the last instar of Drosophila melanogaster that illustrates extensive
basal infoldings, a relatively short path across the narrow cell, and long
microvilli on the apical surface of the cells.
Transport of Substances Through
the Malpighian Tubule Cells
The Rate of Urine Formation by an
Isolated Malpighian Tubule
(from the stick insect Carausius morosus)
A diagram of the Rectal Papillae in
the Rectum of Adult Dipterans
A SEM of the Hemolymph Side of the
Rectum of the Tephritid Fruit Fly
A Cryptonephridial Complex
A Cryptonephridial Complex
Fig. Cross-sectional view of the rectum with cryptonephridial tubules
from the yellow mealworm Tenebrio molitor.
Fig. The countercurrent arrangement of the water-extraction
apparatus of the rectum of the mealworm Tenebrio molitor.
The Insect Excretory System